LOCUS       sequence-183894-        7302 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral gRNA plasmid targeting human PPP2R2D.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7302)
  AUTHORS   McKinley KL, Cheeseman IM
  TITLE     Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the
            Diversity of p53-Dependent Responses to Cell-Cycle Defects.
  JOURNAL   Dev Cell. 2017 Feb 27;40(4):405-420.e2. doi: 
            10.1016/j.devcel.2017.01.012. Epub 2017 Feb 16.
  PUBMED    28216383
REFERENCE   2  (bases 1 to 7302)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.devcel.2017.01.012"; journalName: "Dev Cell"; date: 
            "2017-02-27- 27"; volume: "40"; issue: "4"; pages: "405-420.e2"
COMMENT     Sequence Label: PPP2R2D B8.5 gRNA
FEATURES             Location/Qualifiers
     source          1..7302
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     14..31
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    148..169
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(184..189,190..207,208..214)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    222..238
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     227..249
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     246..262
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     246..262
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     281..301
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        283..301
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        329..555
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             556..736
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    783..908
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1401..1634
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1819..1863
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        2041..2289
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2041..2061
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2212..2231
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2445..2562
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2631..2842
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2868..3467
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2868..2887)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3364..3384
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3483..4071
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3536..3556)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3954..3965)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4143..4376
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4454..4575
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4491..4510)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4545..4564
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4615..4750
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4770..4789)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4771..4789)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4799..4816)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4799..4815)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4808..4830)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4957..5412
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5044..5063)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5254..5275
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5438..5542
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5543..5611,5612..6403)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5761..5780)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6574..7162
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7063..7082
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
       61 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
      121 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
      181 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
      241 tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag
      301 ggaacaaaag ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc
      361 aacatggtaa cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc
      421 cgattggtgg aagtaaggtg gtacgatcgt gccttattag gaaggcaaca gacgggtctg
      481 acatggattg gacgaaccac tgaattgccg cattgcagag atattgtatt taagtgccta
      541 gctcgataca taaacgggtc tctctggtta gaccagatct gagcctggga gctctctggc
      601 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
      661 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
      721 tggaaaatct ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg
      781 agctctctcg acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc
      841 gactggtgag tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg
      901 agagcgtcag tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc
      961 cagggggaaa gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac
     1021 gattcgcagt taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac
     1081 agctacaacc atcccttcag acaggatcag aagaacttag atcattatat aatacagtag
     1141 caaccctcta ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca
     1201 agatagagga agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc
     1261 agacctggag gaggagatat gagggacaat tggagaagtg aattatataa atataaagta
     1321 gtaaaaattg aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga
     1381 gaaaaaagag cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc
     1441 actatgggcg cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata
     1501 gtgcagcagc agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc
     1561 acagtctggg gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag
     1621 gatcaacagc tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg
     1681 ccttggaatg ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg
     1741 atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa
     1801 tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt
     1861 ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata
     1921 gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt
     1981 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccaga
     2041 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
     2101 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
     2161 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
     2221 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
     2281 cgaaacaccg ttcggccacg tcctcgtcga gtttaagagc tatgctggaa acagcatagc
     2341 aagtttaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
     2401 tttgaattct agatcttgag acaaatggca gtattcatcc acaattttaa aagaaaaggg
     2461 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     2521 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
     2581 agcagagatc cactttggcg ccggctcgag tggctccggt gcccgtcagt gggcagagcg
     2641 cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta
     2701 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
     2761 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
     2821 cgggtttgcc gccagaacac aggtgtcgtg acgcgggatc cgccaccatg accgagtaca
     2881 agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg
     2941 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc
     3001 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     3061 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     3121 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     3181 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     3241 tggccaccgt cggagtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     3301 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     3361 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     3421 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaacg cgttaagtcg
     3481 acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     3541 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     3601 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     3661 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     3721 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     3781 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     3841 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     3901 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     3961 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     4021 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg cgtcgacttt
     4081 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg
     4141 actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt gtactgggtc
     4201 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     4261 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     4321 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtac
     4381 gtatagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga aatgaatatc
     4441 agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     4501 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     4561 catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc gcccatcccg
     4621 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4681 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     4741 tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac gcgcgctcac
     4801 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     4861 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     4921 cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa
     4981 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     5041 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     5101 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     5161 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     5221 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     5281 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     5341 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     5401 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5461 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5521 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5581 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5641 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5701 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5761 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     5821 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5881 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5941 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     6001 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     6061 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     6121 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     6181 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     6241 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     6301 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     6361 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6421 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6481 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6541 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6601 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6661 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6721 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6781 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6841 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6901 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6961 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     7021 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     7081 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     7141 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7201 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     7261 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cg
//
